Method and device for preparing sample slide

ABSTRACT

A method for preparing a sample slide, characterized in that a liquid containing a cell floating therein is fixed under an atmosphere for extension wherein an index of dryness as a control parameter in the preparation of a sample slide foe a material to be tasted is adjusted at a level such that the optimum condition is achieved for the preparing of a sample slide.

FIELD OF THE INVENTION

The present invention relates to a method and a device for preparing asample slide of a cell or a chromosome by fixing said analyte on thesample slide. More particularly, the present invention relates to amethod and a device for preparing a sample slide displaying a metaphasein an appropriate shape with a predetermined spreading. The presentinvention is useful in preparing a sample slide of a nucleated cell.

BACKGROUND ART

Generally, a procedure of chromosome inspection may consist from fivestages: cell culture (first step); harvest of cell and preparation ofchromosome sample slide (second step); staining of chromosome bands bydifferential staining technique (third step); microphotography (fourthstep); and karyotype analysis (fifth step). The present invention mainlyfocuses on the harvest of the cell and the preparation of the chromosomesample slide in the second step.

For the harvest of the cell and the preparation of the chromosome sampleslide in the second step, the culture cell obtained in the first step istreated with colcemid to give a metaphase cell. Then, the metaphase cellis subjected to hypotonic treatment and physical shock to destroy thecell membrane and the nuclear membrane, thereby spreading out thechromosome present in the spherical nuclear on a glass slide. Thus,there is a need for preparing a sample slide displaying the metaphase inan appropriate shape with a predetermined spreading. If the sample slidedoes not come out clear, the subsequent processes such as the chromosomeband staining becomes noticeably difficult.

Specifically, according to a conventional technique for preparing achromosome sample slide, a cell-floating fluid containing chromosome ismanually dropped on a slide to spread and fix the chromosome present inthe spherical nuclear on the glass slide. In other words, according tothe conventional technique for preparing a chromosome sample slide, eachof the steps of the fixing technique greatly depends on the skill of thetechnician.

Examples of the manual technique are shown in FIGS. 19 to 22. Accordingto a flame fixing technique shown in FIG. 19, a drop 4 of acell-floating fluid 3 from a pipette 2 is spread out on a sample slide(glass slide) 1. Then, the sample slide 1 is quickly passed through theflame of a burner 5 to set fire to methanol for evaporating the liquidmass from the cell-floating fluid 3, thereby preparing a sample.

According to a steam fixing technique shown in FIG. 20, a sample slide 1prepared by dropping a cell-floating fluid 3 as shown in FIG. 19 isplaced close to steam 8 evaporating from a container 7 heated with aheater 6 so that the liquid mass of the cell-floating fluid 3 isevaporated, thereby preparing a sample.

According to a fixing technique at a high temperature and a highhumidity shown in FIG. 21, a glass slide 1 a is placed in ahigh-temperature and high-humidity environment provided by a wet papertowel 10 heated with a hot plate 9 and a cell-floating fluid 3 isdropped 4 and spread on the glass slide 1 a in the same manner as thetechnique shown in FIG. 19 so that a sample is prepared by evaporatingthe liquid mass of the cell-floating fluid 3.

Furthermore, according to a fixing technique at room temperature bydropping from a high altitude as shown in FIG. 22, a cell-floating fluid3 is dropped 4 from a height of several millimeters or sometimes about1.5 meters to be spread out on a glass slide 1 a seated on a workingtable and the liquid mass of the cell-floating fluid 3 is evaporated,thereby preparing a sample.

The above-described techniques require a certain skill to give ametaphase of chromosome in the cell-floating fluid 3 in an appropriateshape. Moreover, each technique is somehow established upon contingentincidents, rendering it difficult to prepare uniform sample slides.Thus, not anyone is able carry out these techniques. In other words,optimal conditions for obtaining fixation suitable as an analyte are notruled for these techniques and therefore fixation depends on empiricalknowledge of the practitioner.

Needless to say, according to the above-described conventionalskill-required techniques for preparing a sample slide, it is difficultfor an inexperienced practitioner to prepare a sample slide of ametaphase chromosome in an appropriate shape as a sample. In addition,these conventional techniques are poor in reproducibility of theoperation conditions and in quality stability due to manual proceduresby the practitioner.

In view of the above-described problems, the present invention has anobjective of providing a method feasible for everyone for preparing asample slide suitable as an analyte by controlling dryness, a parameterinvolved in the sample slide preparing method and an objective ofproviding a device for preparing a sample slide with stable quality andfurthermore allowing mass production of the sample slide.

DISCLOSURE OF THE INVENTION

The present inventors have gone through studies in order to achieve theabove-mentioned objectives and found that a metaphase in an appropriateshape can be prepared by drying a liquid analyte in an appropriatedrying environment, thereby accomplishing the present invention.

According to the method for preparing a sample slide of the invention, acell-floating fluid is fixed in a spreading environment where dryness, acontrol parameter of sample slide preparation, is adjusted so as toobtain optimal conditions for preparing the sample slide.

Furthermore, the dryness is obtained by providing a temperature andhumidity sensors in the spreading environment for the cell-floatingfluid to measure the temperature and the humidity within the spreadingenvironment, obtaining a saturated moisture value at the saidtemperature and, based on this saturated moisture value and the actualabsolute humidity, calculating according to the following equation:Dryness [Idry]=Saturated moisture [Ws]−Absolute humidity [AbsH].

The device for preparing the sample slide of the invention where ananalyte of a cell or a chromosome is fixed thereon is provided with amechanism for controlling the dryness of the environment for spreadingthe metaphase analyte on the sample slide.

The mechanism for controlling the dryness measures the temperature andthe humidity in the cell-floating fluid spreading environment with thetemperature and humidity sensors arranged in the environment, obtain thesaturated moisture value at the said temperature and, calculate thedryness based on this saturated moisture value and the actual absolutehumidity.

The device for preparing the sample slide is further provided with amechanism for preparing a liquid analyte for obtaining a liquid analyteto be added onto the sample slide dropwise.

According to the method and the device for preparing a sample slide,anyone can prepare a sample slide with optimal cell or chromosome fixingby controlling, among the control parameters for optimal fixing of theanalyte on the sample slide, the dryness of the dropped cell-floatingfluid.

The present invention further has an advantage of allowing automatedpreparation of a quality stable sample slide, enabling mass-productionthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing measurement of dryness according to amethod for preparing a sample slide of the invention;

FIG. 2 is a schematic view illustrating the relationship between thedryness and the spreading of cell;

FIG. 3 is a schematic view illustrating the spreading of cell when thedryness is high;

FIG. 4 is an exemplary sample slide obtained in the case of FIG. 3;

FIG. 5 is a schematic view illustrating the spreading of cell when thedryness is optimal;

FIG. 6 is an exemplary sample slide obtained in the case of FIG. 5;

FIG. 7 is a schematic view illustrating the spreading of cell when thedryness is low;

FIG. 8 is a cross-sectional view showing a first embodiment of thedevice for preparing a sample slide of the invention;

FIG. 9 is a plan view showing a substantial part of a second embodimentof the device for preparing a sample slide of the invention;

FIG. 10 is a cross-sectional view of the device for preparing a sampleslide shown in FIG. 9 cut along line X-X;

FIG. 11 is a cross-sectional view of the device for preparing a sampleslide shown in FIG. 9 cut along line XI-XI;

FIG. 12 is a cross-sectional view showing a third embodiment of thedevice for preparing a sample slide of the invention;

FIG. 13 is a plan view showing a substantial part of the device forpreparing a sample slide shown in FIG. 12;

FIG. 14 is a schematic view illustrating a structure of a centrifugeaccording to the third embodiment of the device for preparing a sampleslide of the invention;

FIG. 15 is a schematic view illustrating a structure of an automateddryness controller according to the third embodiment of the device forpreparing a sample slide of the invention;

FIG. 16 is a flowchart showing a procedure at a first stage forpreparing a glass sample slide with the sample slide preparing device ofthe third embodiment;

FIG. 17 is a flowchart showing a procedure at a second stage forpreparing a glass sample slide with the sample slide preparing device ofthe third embodiment;

FIG. 18 is a flowchart showing a procedure at a third stage forpreparing a glass sample slide with the sample slide preparing device ofthe third embodiment;

FIG. 19 is a schematic view showing a conventional method for fixing acell-floating fluid (flame fixing technique);

FIG. 20 is a schematic view showing a conventional method for fixing acell-floating fluid (steam fixing technique);

FIG. 21 is a schematic view showing a conventional method for fixing acell-floating fluid (hot plate fixing technique); and

FIG. 22 is a schematic view showing a conventional method for fixing acell-floating fluid (naturally dried fixing technique).

BEST MODE FOR CARRYING OUT THE INVENTION

First, a method for preparing a sample slide of the invention will bedescribed.

The method for preparing a sample slide of the invention ischaracterized in that dryness in an enclosed space for spreading acell-floating fluid as an analyte is controlled.

In order to control the dryness according to the present embodiment, thedryness needs to be determined. Determination of the dryness hereinrefers to determination of how much moisture that ambient atmosphere cantake up. This can be calculated based on an actual moisture contentunder that environment and a saturated moisture content.

The dryness can be calculated based on temperature and absolute humidityas follows.Dryness [Idry]=Saturated moisture content [Ws]−Absolute humidity [AbsH].Dryness [Idry]: Dryness (g/m³)Saturated moisture content [Ws]: Saturated moisture content attemperature T° C. (g/m³)Absolute humidity [AbsH]: Absolute humidity (g/m³)

With reference to FIG. 1, according to the present embodiment, atemperature sensor 11 is arranged at a site where the temperature of thesurface of the sample slide 1 where a cell-floating fluid is dropped andthe ambient temperature in the environment for spreading thecell-floating fluid can properly be measured. A humidity sensor 12 isplaced in the spreading environment. A detection circuit (a saturatedmoisture content detecting unit) 13 outputs a saturated moisture contentat a temperature detected by the temperature sensor 11. The output fromthe detection circuit 13 and the absolute humidity actually measured bythe humidity sensor 12 are used such that a control circuit (a drynesscalculating unit) 14 outputs dryness according to the above-mentionedequation and the result is displayed on a display unit 16. In FIG. 1,the temperature sensor and the humidity sensor are illustrated as adryness sensor 15 having both of the functions.

The dryness is based on two factors, i.e., an energy put upon the fluidand how much of the vaporized fluid the ambient atmosphere can take up.

The energy that rises upon preparing the sample slide refers to heatenergy. In order to give a constant heat energy, the surface temperatureof the glass slide 1 a (where the cell-floating fluid 3 is dropped 4)needs to be kept constant. Therefore, the thermostatic property of thesurface of the glass slide is an important factor.

The amount of fluid that can be taken up in the atmosphere of theenvironment is determined according to the vapor pressure of thevaporized fluid. When mixed gases are used, the vapor pressure changesaccording to the proportion of the gases in the mixture. Thecell-floating fluid 3 used for the preparation of the sample slideaccording to the present embodiment is Carnoy's fixtative, a mixture ofacetic acid and methanol. Both liquids evaporate. According to thepresent embodiment, the dryness is controlled by controlling the amountof vapor in the vaporizing environment to control the vaporized amountof the Carnoy's fixtative.

By controlling the amount of vapor in the environment for spreading thecell-floating fluid 3, an environment with a humidity optimal forpreparing a sample slide is produced. By equilibrating vapor pressuresof methanol/acetatic acid and water, dryness of the droppedcell-floating fluid is kept optimal. As a result, a sample slide 1 withan optimal cell or chromosome fixing is obtained.

Specifically, while a predetermined amount of the cell-floating fluid 3is supplied, namely a predetermined amount of an analyte is dropped, anda predetermined concentration of acetic acid in the Carnoy's fixtativeis used for fixing the analyte, the saturated moisture value output atthis temperature and the actual humidity are used to calculate thedryness according to the above-mentioned equation. The saturatedmoisture value at the calculated temperature is calculated for variouscombinations of the supply amount of the cell-floating fluid 3 and theconcentration of the acetic acid in the Carnoy's fixtative so that thesevalues are stored in a memory such as ROM (not shown) for theabove-mentioned saturated moisture value detecting unit 13 to be able torefer to the memory.

For example, when the amount of the cell-floating fluid 3 supply isfixed to 0.04 ml while the acetic acid concentration in the Carnoy'sfluid is fixed to 3:1 (methanol: acetic acid), the dryness at atemperature of 30° C. is 5-10 g/m², preferably 7 g/m². Thus, the vaporamount in the vaporized environment is controlled to give this dryness.

Hereinafter, the relationship between the dryness and the spreading ofthe cell will be described with reference to FIGS. 2 to 7. The lower thedryness is, the wider the cell-floating 3 spreads. On the contrary,higher dryness keeps the cell-floating fluid 3 to stay smaller.

The cells after being dropped on the slide travels in various directionalong with the fluid and adhere on the surface of the glass slide 1 a asthe Carnoy's fluid evaporates. As the cell adhered on the glass slide 1spreads, first the cell membrane is broken due to the surface tension ofthe Carnoy's fluid and the intracellular fluid containing the chromosomeand the like begins to flow.

However, at the size level of a cell, Reynolds number (=(inertialforce/viscous power)=(density/viscosity)×(length)×(rate)) is very small,and thus the viscosity of the intracellular fluid becomes high in thearea so the flow rate is very low.

The intracellular fluid is believed to be a fluid with a great viscosity(for example, internal viscosity of a white blood cell is 13 Pa/s). Whenthe flow rate of the intracellular fluid is slower than the descendingrate of the surface level of the dropped cell-floating fluid, the cellis fixed with small spreading as shown in FIGS. 3 and 4. When the dryingrate is optimal, optimal spreading as shown in FIGS. 5 and 6 can beachieved. When the drying rate is slow (dryness is low), the chromosomeas an internal substance of the cell spreads out too much such that itno longer stays at one site but is dispersed, and thus the resultingsample is not suitable for genome analysis or the like.

According to the method for preparing the sample slide of the presentembodiment, among the control parameters involved in optimal fixing ofthe cell or chromosome onto a sample slide, dryness of the cell-floatingfluid 3 dropped 4 is particularly controlled. As a result, anyone canprepare a sample slide with an optimal cell or chromosome fixing asshown in FIGS. 5 and 6.

Next, the above-described method for preparing a sample slide will bedescribed more specifically.

First, the cell-floating fluid 3 as an analyte is cultured beforehandfollowed by hypotonic treatment and Carnoy's fixative treatment. Asdescribed above, the cell-floating fluid 3 used for preparing a sampleslide of the present embodiment is a Carnoy's fixative which is amixture solution of acetic acid and methanol.

On the other hand, a temperature sensor 11 and a humidity sensor 12placed in the environment for spreading the Carnoy's fixative, or thecell-floating fluid 3 are used to measure the temperature and thehumidity in the spreading environment. A saturated moisture valuedetecting unit 13 outputs a saturated moisture at that temperature basedon the amount of cell-floating fluid 3 supply, i.e., the amount ofanalyte dropped, and the concentration of the acetic acid in theCarnoy's fixative added upon preparing the analyte. Then, a drynesscalculating unit 14 calculates the dryness according to theabove-mentioned equation using the output of the saturated moisturevalue and the actual absolute humidity. A display unit 16 constantlyoutputs this dryness.

This output dryness in the spreading environment is adjusted to a valueoptimal for fixing the analyte. Specifically, the spreading environmentis exposed to outside to release the humidity from the environment tolower the humidity inside the spreading environment. Alternatively, thespreading environment is humidified, or in some cases heated or cooledto alter the saturated moisture value at that temperature to attain themost optimal dryness.

In this adjusted environment, a predetermined amount of thecell-floating fluid 3 is dropped 4 on the surface of the sample slide 1placed in an enclosed space as the spreading environment and left for apredetermined period of time. The dry atmosphere inside the enclosedspace allows optimal spreading and fixing of the analyte. Next, a devicefor carrying out the method for preparing a sample slide will bedescribed specifically.

FIG. 8 is a cross-sectional view showing a substantial fundamentalstructure of a first embodiment of the device for preparing a sampleslide of the invention.

The main body of the device 17 for preparing a sample slide of thepresent embodiment is provided with a thermostatic block 18 on which aglass slide 1 a is mounted and which provides a stable amount of heat tothe glass slide 1 a, a face place 19 for supporting the thermostaticblock 18, a spreading space cover 20 for forming an enclosed space abovethe face plate 19 as an environment for spreading a metaphase analyte onthe glass slide 1 a, and a heating unit 21 for appropriately controllingthe temperature of the cell-floating fluid 3 as an analyte via thethermostatic block 18 supported by the face place 19.

According to the present embodiment, the thermostatic block 18 isprovided with a rectangular thermostatic block member 18 a and a heattransferring member 18 b having a plurality of plate fins aligned inparallel and generally vertical to and integral with the bottom surfaceof the thermostatic block member 18 a. In the present embodiment, theentire thermostatic block 18 is made of a metal with good heatconductivity, i.e., aluminum, in a size sufficient to provide asatisfactory heat capacity. The thermostatic block member 18 a issupported by the face place 19 such that the upper surface thereof isgenerally horizontally exposed. One or more slide guides 22 are providedon the upper surface of the thermostatic block member 18 a. The slideguides 22 guide one or more glass slides 1 a to be mounted.

The face plate 19 is provided with a first humidity adjusting fin 23which allows communication between inside and outside a storage tankdescribed later. A dryness sensor 15 is provided on the face plate 19 inthe vicinity of the thermal block member 18 a, which sensor 15calculates dryness inside the enclosed space for spreading a metaphaseanalyte on the glass slide 1 a. The dryness sensor 15 includes sensorsthat can measure the temperature and the humidity in the enclosed space,which are data necessary for calculating the dryness.

The spreading space cover 20 includes surrounding walls (side panels) 20a and a ceiling (top panel) 20 b which, together with the face plate 19(bottom panel) supporting the thermostatic block 18, form the enclosedspace. The ceiling 20 b of the spreading space cover 20 is provided witha second humidity adjusting fin 24 that allows communication betweeninside and outside the sample slide preparing device, at a part opposingthe first humidity adjusting fin 23 in an enclosed state of the cover20. The ceiling 20 b is further provided with a pipette 25 with its tipbeing arranged such that the cell-floating fluid 3 as an analyte can bedropped 4 on the center part of the glass slide 1 a mounted on thethermostatic block 18.

The heating member 21 is arranged below the face plate 19 and forms ahomeothermal water bath with a storage tank 26 in which the thermostaticblock 18 is partially immersed and a heater 27 for heating the waterstored in the tank 26 to an appropriate temperature. The surroundingwalls 26 a and the bottom 26 b of the storage tank 26 are made of a heatinsulating material. At the bottom of the surrounding wall 26 a of thestorage tank 26, a discharge cock 28 is provided for discharging thestored water. According to the present embodiment, the heater 27 canmanually be switched on or off. Alternatively, a dryness controllingunit 30 described later may automatically control the switching andtiming of an electric current to control heating. The face plate 19 isarranged to cover the entire upper opening of the storage tank 26. Thelower ends of the plate fins of the heat transferring member 18 b areimmersed in water 29 at an appropriate temperature stored in the storagetank 26.

The device 17 for preparing the sample slide of the present embodiment,is further provided with peripheral equipments such as a pumpcontrolling unit (not shown), the saturated moisture value detectingunit 13, the dryness calculating unit 14, a MANUAL/AUTOMATIC controlswitch (not shown) and a dryness controlling unit (not shown) that takesover controls when the control switch is switched to AUTOMATIC.

The supply pump (not shown) of the pump controlling unit supplies apredetermined amount of a cell-floating fluid 3 that has been culturedand subjected to hypotonic treatment, dropwise to the surface of thesample slide 1 via a carrier pipe (not shown) and the drop pipette 25arranged in the enclosed space.

The saturated moisture value detecting unit 13 detects the saturatedmoisture value at the temperature measured by the temperature sensor 11by referring to the memory. The dryness calculating unit 14 outputs anddisplays on the display unit 16 the dryness in the spreading environmentcalculated according to the above-mentioned equation based on thesaturated moisture value and the value obtained by the humidity sensor12. Moreover, when AUTOMATIC is selected by the MANUAL/AUTOMATIC controlswitch, the dryness controlling unit is initiated.

The dryness controlling unit refers to the resulting dryness calculatedby the dryness calculating unit 14, controls switching of the heater 27of the homeothermal water bath and the timer 27 a and, when it isprovided with a mechanism for automatically opening and closing thefirst humidity adjusting fin 23 and the second humidity adjusting fin24, controls the opening and the closing thereof so that optimal drynessis obtained in the analyte spreading environment.

Hereinafter, a method for preparing a sample slide using the sampleslide preparing device of the present embodiment will be describedbriefly.

First, a glass slide 1 a is placed on the upper surface of the heatedthermostatic block member 18 a as guided by the slide guide 22. Then,the spreading space cover 20 is closed to enclose the environment forspreading a metaphase analyte. At this point, a predetermined amount ofa cell-floating fluid 3 is dropped 4 on the glass slide 1 a by thesupply pump of the pump controlling unit via the drop pipette 25 tospread a metaphase analyte.

In the enclosed space as the spreading environment, the water stored inthe storage tank 26 is heated to an appropriate temperature 29 byrunning a current to the heater 27 arranged in the storage tank 26. Thesurface of the thermostatic block member 18 a is heated to anappropriate temperature via the plate fins of the heat transferringmember 18 b where the lower ends of the plate fins are immersed in thewater at the appropriate temperature 29. According to the presentembodiment, the dryness calculated by the saturated moisture valuedetecting unit 13 and the dryness calculating unit 14 and displayed onthe display unit 16 is adjusted to a humidity optimal for spreading theanalyte within the enclosed environment. Specifically, the opening orclosing of the first humidity adjusting fin 23 is controlled to sendexcess vapor from the water at the appropriate temperature 29 fillingthe storage tank 26 to the spreading environment to keep thepredetermined dryness that is constantly under control. Alternatively,the opening or closing of the second humidity adjusting fin 24 iscontrolled to expose the sample slide preparing device 1 to outside withlower humidity to obtain desirable dryness. If necessary, the heater 27is switched on to adjust the temperature of the water 29 for vaporproduction or humidification. When AUTOMATIC is selected by theMANUAL/AUTOMATIC control switch, the opening or closing of the first andsecond humidity adjusting fins 23 and 24 is automatically controlled viathe dryness controlling unit 30 as described above.

Thus, the analyte on the sample slide 1 is dried by being exposed to theadjusted dry atmosphere in the spreading environment.

By controlling the dryness as the parameter involved in preparing thesample slide 1, the metaphase analyte can be formed into an appropriateshape and anyone can prepare an appropriate sample slide 1 with stablequality.

FIGS. 9 to 11 are schematic views showing fundamental structure of asecond embodiment of a device for preparing a sample slide of theinvention.

The sample slide preparing device 31 of the present embodiment isparticularly distinct from the device of the first embodiment in that acommercially-available water bath 32 is used instead of the storage tank26 as the homeothermal water bath for controlling the temperature of thethermostatic block 18. Hereinafter, parts of the structure that differfrom those of the sample slide preparing device 17 described above willbe described briefly. Parts that are identical to those of the sampleslide preparing device 17 of the first embodiment will be denoted by thesame reference numbers and the descriptions thereof are omitted.

The sample slide preparing device 31 according to the present embodimentis provided with a spreading device 33 for spreading an analyte on aglass slide 1 a and the water bath 32 mentioned above.

The spreading device 33 includes a rectangular chassis 33 a that coversa thermostatic block 18 in a strip-like arrangement at the upper centerpart between a pair of side walls of the chassis 33 a, a top board 34integrally formed with the side wall of the chassis 33 a so as to fillthe gap between the thermostatic block 18 and the chassis 33 a at oneside of the thermostatic block 18, and a first humidity adjusting fin 23made of a single board supported at an axis by a rotation shaft 35between the thermostatic block 18 and the chassis 33 a at the other sideof the thermostatic block 18.

The chassis 33 a is hinged to a spreading space cover 20 that can freelybe opened and closed to provide an enclosed space for spreading ananalyte above the thermostatic block 18, the top board 34 and the firsthumidity adjusting fin 23.

A plurality of glass slides 1 a can be aligned on the upper surface ofthe thermostatic block 18 with slide guides 22 arranged therebetween asseparators for preventing the cell-floating fluid 3 dropped on the glassslide 1 a from invading the adjacent glass slide 1 a.

To the bottom surface of the thermostatic block 18, a heat transferringmember 18 b is secured which stands in the water bath 32 as feet of thespreading device 33 and functions as heat transfer fins for providingstable heat to the glass slides 1 a from the water 29 at an appropriatetemperature stored in the water bath 32 via the thermostatic block 18.

The top board 34 is provided with a dryness sensor 15 identical to thatof the first embodiment. Both ends of the rotation shaft 35 supportingthe first humidity adjusting fin 23 stick out of the opposing side wallsof the chassis 33 a and are provided with adjustment knobs 36. Theadjustment knobs 36 can be grabbed and rotated so that the firsthumidity adjusting fin 32 (

23?) can be rotated manually.

A second humidity adjusting fin 24 for communicating inside and outsidethe sample slide preparing device 31 is slidably arranged at a part ofthe ceiling 20 b of the spreading space cover 20 so as to oppose thefirst humidity adjusting fin 23 when the spreading space cover 20 is ina closed state. The ceiling 20 b is also provided with a drop pipette 25such that a cell-floating fluid 3 as an analyte can be dropped 4 on thecenter of each glass slide 1 a aligned on the surface of thethermostatic block 18.

The water bath 32 is filled with water 29 at an appropriate temperaturewhich is kept at a constant temperature with a heater for adjusting thetemperature (not shown).

The lower end of the heat transferring member 18 b, or the heat transferfins and the entire lower circumference of the chassis 33 a of thespreading device 33 are placed in the water at the appropriatetemperature in the water bath 32.

Similar to the first embodiment, such sample slide preparing device 31is able to prepare a sample slide 1 with a desirable spreading of theanalyte. Since the sample slide preparing device 31 of the presentembodiment utilizes a commercially available water bath 32, the costthereof can be lowered. In addition, the device can be downsized evenwith a plurality of glass slides 1 a in an alignment arrangement.

A sample slide preparing device 40 shown in FIGS. 12 and 13 performscell harvest by repeating hypotonic treatment and Carnoy's fixationtreatment using a centrifuge 44, and automatically prepares a sampleslide of a cell or a chromosome in a spreading environment adjusted todryness of optimal fixing conditions as described above. Parts identicalto those of the sample slide preparing device 17 of the first embodimentwill be denoted by identical reference numbers and the descriptionsthereof are omitted.

The sample slide preparing device 40 according to the present embodimentis provided with a thermostatic block 18 for fixing an analyte at oneside within a rectangular main body 41. A slide cassette 1 b including aset of glass slides 1 a is supplied on the upper surface of thethermostatic block 18 from the adjacent glass slide supplying cassetteunit 42. A liquid analyte preparing mechanism 43 for obtaining a liquidanalyte to be dropped on a sample slide 1 is arranged at the back of thethermostatic block 18. The liquid analyte preparing mechanism 43 mainlyconsists of a centrifuge 44 and a XYZ-direction movable pipettingmechanism 45 for dropping the liquid analyte on the sample slide 1.

Two parallel elevated rails 46 longitudinally run along both sides ofthe thermostatic block 18 and the centrifuge 44. The XYZ-directionmovable pipetting mechanism 45 can run along the elevated rails 46 viarollers 48 and includes a pipette supporting unit 50 which can run alonga crossing rail 47 bridging between the elevated rails 46 via rollers49. The pipette supporting unit 50 is provided with a drop pipette 25that is free to move up and down.

The centrifuge 44 will be described with reference to FIG. 14.

The centrifuge 44 includes a rotation member 53 secured to an open endof a rotation axis 52 of a centrifuge rotating mechanism 51 such as amotor. Six oscillating buckets 54 are arranged evenly spaced apart alongthe outer circumference of the rotation member 53 having the rotationaxis 52 at the center. Each of the oscillating buckets 54 is providedwith a tube 55 rotatable around the center axis thereof. The centrifugeposition detecting mechanism 56 detects that each of the tubes 55 hashalted at a predetermined halt position. Specifically, halt positionmarks (not shown) provided along an outer circumference of a sensor disk57 a secured to the rotation axis 52 are detected with a sensor 57.

As shown in FIG. 14, a hypotonic solution pipette 58 for injecting apredetermined amount of liquid reagent (hypotonic solution or Carnoy'sfixative) into the tubes 55 held by the oscillating buckets 54 and adischarge pipette 59 for discharging a predetermined amount of liquidfrom the tubes 55 are provided around the centrifuge 44. As shown inFIG. 14, a Carnoy's fixative pipette 60 is also provided at the sameposition as the hypotonic solution pipette 58. An agitating mechanism 63is arranged below the hypotonic solution pipette 58, which mechanism 63has a driving member (not shown) including an up-and-down agitatingmechanism 62 that holds the lower end of each of the halting tubes 55with an upwardly extending gripper 61 to move each tube 55 up and downto allow axial rotation of each tube 55 in clockwise and anti-clockwisedirections with respect to the oscillating buckets 54. The hypotonicsolution pipette 58 is supplied with a hypotonic solution from ahypotonic reagent bottle 64 with a delivery pump 65. The Carnoy'sfixative pipette 60 is supplied with acetic acid from an acetic acidbottle 66 and methanol from a methanol bottle 67 at a predeterminedproportion (1:3 in the present embodiment) with delivery pumps 68 and69, respectively, via a mixer 70 where the two substances are mixed. Thedrainage in the tubes 55 is suctioned with the discharge pipette 59 bythe discharge pump 74 and discharged into a drainage tank 71. The amountof drainage in the tubes 55 can be detected with a drainage leveldetector 72.

The centrifuge rotating mechanism 51, the centrifuge position detectingmechanism 56, the agitating mechanism 63 and the delivery pumps 65, 68and 69 are each connected to and controlled by a controller 73 of thesample slide preparing device 40.

The sample slide preparing device 40 of the present embodiment is alsoprovided with an automatic dryness controller 80 including theabove-described dryness calculating unit 14 and the dryness controllingunit 30 as schematically shown in FIG. 15.

As shown in the figure, the automatic controller 80 of the presentembodiment includes a temperature control loop and a dryness controlloop for the thermostatic block 18 for mounting a glass slide 1 a. Thetemperature control loop includes a temperature sensor 11 arranged inthe vicinity of the thremostatic block 18, a temperature adjustingmechanism 83 having a heater 81 and a Peltier cooling element 82 and atemperature control calculating circuit (temperature control calculatingunit) 84. The dryness control loop includes the temperature control loopas well as a humidifying water tank 85, a heater 27, a humidity sensor12, the dryness calculating unit 14 having a saturated moisture valuedetecting unit for determining a saturated moisture value and thedryness controlling unit 30 for controlling a power supply to theheaters 81 and 27 and the temperature adjusting mechanism 83.

Since the automatic dryness adjusting device 80 of the sample slidepreparing device 40 of the present embodiment allows adjustment of thetemperature and the dryness in the environment for spreading an analyte,sample preparation conditions suitable for various nucleated cellsdiffering in viscosities of their intracellular fluids can be provided.

Hereinafter, operations of the present embodiment will be described withreference to FIGS. 16 to 18. The environment for spreading an analyte inthe sample slide preparing device of the present embodiment is adjustedaccording to the above-described method and thus the descriptionsthereof are omitted.

FIGS. 16, 17 and 18 are flowcharts illustrating processes at first,second and third stages, respectively, for preparing a sample glassslide with the sample slide preparing device of the present invention.

First, at the preparatory stage, cultured cell-floating fluids aretransferred into the six tubes 55 each held with the oscillating buckets54 of the centrifuge 44.

Then, the centrifuge 44 is driven at 1,300 rpm for 10 minutes. With theabove-described centrifuge position detecting device 56, one of thetubes 55 (hereinafter, operations will mainly be described for thissingle tube 55) is halted at a position of the discharge pipette 59(Step ST1).

The discharge pipette 59 is inserted into the halting tube 55 from abovewithout touching the inner wall of the tube 55 to a predetermined depthof the supernatant as detected with the drainage level detector 72.Subsequently, the discharge pump 74 is driven to suction the supernatant(about 5 ml) from the tube 55 and to discharge into the drainage tank 71(Step ST2).

Once the extraction of the supernatant is completed, the dischargepipette 59 is drawn out of the tube 55. Then, the centrifuge rotatingmechanism 51 is driven to transfer the tube 55 to the position of thehypotonic solution pipette 58 where a predetermined amount (5 ml) of ahypotonic solution is injected into the tube 55 from the hypotonicsolution pipette 58 by driving the delivery pump 65. Thereafter, thetube 55 is subjected to eccentric agitation in a pestle-like movement(Step ST3).

The tubes 55 are sequentially transferred at one halt position at a timein a rotation direction of the rotating member 53 to repeat theprocesses of Steps ST2 and ST3.

Then, the rotating member 53 is stopped for about 15 minutes while thetemperature is kept at 37° C. for hypotonic treatment (Step ST4).

Thereafter, all of the tubes 55 are sequentially subjected to secondagitation at the position of the hypotonic solution pipette 58 (StepST5).

The tubes 55 after the second agitation are subjected to hypotonictreatment as in Step ST4 (Step ST6).

At a position of the hypotonic solution pipette 58, a predeterminedamount (0.5 ml) of Carnoy's fixative is injected for the first time fromthe Carnoy's fixative pipette 60 by driving the delivery pumps 66 and67. Thereafter, the tube 55 is subjected to eccentric agitation in apestle-like movement (Step ST7).

The tubes 55 are sequentially transferred at one halt position at atime; in a rotation direction of the rotating member 53 to repeat theprocess of Step ST7.

Then, the centrifuge 44 is driven at 1,300 rpm for 10 minutes (StepST8).

Next, as shown in FIG. 17, when the centrifuge 44 is stopped, thedischarge pipette 59 is inserted into the halting tube 55 from abovewithout touching the inner wall of the tube 55 to a predetermined depthof the supernatant as detected with the drainage level detector 72.Subsequently, the discharge pump 74 is driven to suction the supernatantfrom the tube 55 and to discharge into the drainage tank 71 (Step ST9).

Once the extraction of the supernatant is completed, the dischargepipette 59 is drawn out of the tube 55. Then, the centrifuge rotatingmechanism 51 is driven to transfer the tube 55 to the position of thehypotonic solution pipette 58 so as to inject a predetermined amount (3ml) of the Carnoy's fixative for the second time into the tube 55 fromthe Carnoy's fixative pipette 60 by driving the delivery pumps 66 and 67as in Step ST7. Thereafter, the tube 55 is subjected to eccentricagitation in a pestle-like movement (Step ST10).

The tubes 55 are sequentially transferred at one halt position at a timein a rotation direction of the rotating member 53 to repeat theprocesses of Steps ST9 and ST10.

When all of the tubes 55 have completed agitation, the centrifuge 44 isdriven at 1,300 rpm for 6 minutes (Step ST11).

Once the centrifuge 44 is stopped, each of the tubes 55 halted at theposition of the discharge pipette 59 is sequentially subjected to theprocesses of Steps ST9 to ST11 for three to four times (Step ST12). Atthe final round, the amount of Carnoy's fixative injected at Step ST10is made 1.5 ml, and the tube 55 is subjected to eccentric agitation in apestle-like movement (Step ST10′). Thereafter, the procedure is pausedfor visual inspection after which 1.5 ml of Carnoy's fixative isinjected again followed by eccentric agitation as described for StepST10 (Step ST10″). Next, the operation proceeds to Step ST11.

After the final centrifugation, the operation proceeds to FIG. 18.

At Step ST13 in FIG. 18, supernatant in each of the tubes 55sequentially halting at the position of discharge pipette 59 issuctioned as in Steps ST9 and ST10.

Then, Carnoy's fixative for adjusting the cell-floating fluid isinjected for the last time into the tube 55 halting at the position ofhypotonic pipette 58 from the Carnoy's fixative pipette 60 by drivingthe delivery pumps 66 and 67. Thereafter, the tube 55 is subjected toeccentric agitation in a pestle-like movement (Step ST14). Then, thedrop pipette 25 is inserted in the tube 55 by driving the XYZ-directionmovable pipetting mechanism 45 so as to repeat suctioning anddischarging of 0.1 to 1 ml of the cell-floating fluid 3 in the tube 55as a tapping process (Step ST15).

The supply pump 33 is drivin to collect the cell-floating fluid 3 intothe drop pipette 25 (Step ST16). Then, the XYZ-direction movablepipetting mechanism 45 is driven to draw out the drop pipette 25 of thetube 55. Subsequently, the XYZ-direction movable pipetting mechanism 45is driven to transfer the drop pipette 25 above the spot position of thesample slide 1 and again driven to transfer the tip of the drop pipette25 immediately above the sample slide 1. The supply pump 33 is driven todrop necessary drops of the cell-floating fluid 3 from the drop pipette25 to the glass slide 19 (Step ST17).

For each of the spreading sample, the procedure from Step ST13 to Stl7are repeated for every tubes 55. Until all of the cell spreadings on theglass slides 1 a from a single tube 55 are fixed, other samples are keptto stand still.

Thereafter, according to the method described for the embodimentreferring to FIG. 1, the cell-floating fluid 3 in a liquid form on thesample slide 1 is well dried with the thermostatic block 18 arranged inthe above-described sample slide preparing device, thereby preparing ametaphase with an appropriate shape.

According to the present embodiment, an accurate dropping amount of aliquid analyte, a cell-floating fluid 3 can be dropped on a sample slide1 with a use of an accurate supply pump while keeping the acetic acidconcentration of the Carnoy's fixative constant. Thus, these twoparameters can be fixed to constant values.

The present invention is not limited to the above-described embodimentsand various modification can be made according to necessity.

1. A method for preparing a sample slide, comprising: a cell-floatingfluid is fixed in a spreading environment where dryness, a controlparameter of sample slide preparation, is adjusted so as to obtainoptimal conditions for preparing the sample slide.
 2. The method forpreparing a sample slide according to claim 1, wherein the dryness isobtained by providing a temperature and humidity sensors in thespreading environment for the cell-floating fluid to measure thetemperature and the humidity within the spreading environment, obtaininga saturated moisture value at the said temperature and, based on thissaturated moisture value and the actual absolute humidity, calculatingaccording to the following equation:Dryness (Idry)=Saturated moisture (Ws)−Absolute humidity (AbsH).
 3. Adevice for preparing a sample slide, comprising: an analyte of a cell ora chromosome is fixed thereon is provided with a mechanism forcontrolling the dryness of the environment for spreading the metaphaseanalyte on the sample slide.
 4. The device preparing a sample slideaccording to claim 3, wherein the mechanism for controlling the drynessmeasures the temperature and the humidity in the cell-floating fluidspreading environment with the temperature and humidity sensors arrangedin the environment, obtain the saturated moisture value at the saidtemperature and, calculate the dryness based on this saturated moisturevalue and the actual absolute humidity.
 5. The device for preparing asample slide according to claim 3 or 4, further provided with amechanism for preparing a liquid analyte for obtaining a liquid analyteto be added onto the sample slide dropwise.
 6. A device for preparing asample slide comprising: a thermostatic block including a thermostaticblock member for receiving a glass slide thereon for spreading amethaphase analyte and providing a stable heat and a heat transferringmember provided on the bottom surface of the thermostatic block member;a heating unit including a storage tank for storing water for immersinga part of the heat transferring member and a heater for heating thewater stored in the storage tank to an appropriate temperature; a faceplate arranged to cover the entire opening of the storage tank, tosupport the thermostatic block member such that the upper surfacethereof is exposed generally horizontally, provided with a temperaturesensor and a humidity sensor in the vicinity of the thermostatic blockmember for measuring dryness in an enclosed space for spreading themetaphase analyte and provided with a first humidity adjusting fin thatallows communication between the spaces separated by said face place; asecond humidity adjusting fin for allowing communication between insideand outside the sample slide preparing device provided with surroundingwalls and ceiling that form the enclosed space with the face plate asthe bottom plate; and a spreading space cover provided with a droppipette for dropping a cell-floating fluid as an analyte on the glassslide mounted on the thermostatic block.
 7. A device for preparing asample slide, comprising: a rectangular chassis; a thermostatic blockmember for providing stable heat, provided in a strip-like arrangementat an upper center part between a pair of side walls of the chassis; aheat transferring member made from heat transferring fins arranged atthe bottom surface of the thermostatic block member and to stand in awater bath as a part of a heating member, the heat transferring memberproviding stable heat from water at an appropriate temperature stored inthe water bath to a glass slide via the thermostatic block; a top boardprovided with a temperature sensor and a humidity sensor for measuringdryness in an enclosed space for spreading a metaphase analyte on theglass slide, the top board integrally formed adjacent to the side wallsof the chassis so as to fill in the gap between the thermostatic blockand the chassis formed on one side of the thermostatic block member; afirst humidity adjusting fin that can be opened and closed, provided atthe gap between the thermostatic block and the chassis formed on theother side of the thermostatic block member; a spreading space coverincluding surrounding walls and ceiling which form an enclosed spacewith the thermostatic block member, the top board and the first humidityadjusting fin, the spreading space cover provided with a second humidityadjusting fin that allows communication between inside and outside thesample slide preparing device and a drop pipette for dropping acell-floating fluid as an analyte on a glass slide mounted on thethermostatic block; and a heating unit including the water bath forstoring water at an appropriate temperature for immersing a part of theheat transferring member and a lower part of the entire circumference ofthe chassis, and a heater for heating the water stored in the water bathto the appropriate temperature.
 8. A device for preparing a sample slideaccording to claim 6 or 7, further comprising: a centrifuge including acentrifuge rotating mechanism, tubes held by a plurality of oscillatingbuckets arranged on the centrifuge rotating mechanism, one or moresupplying pipettes for injecting a liquid reagent into the tubes, adelivery pump for delivering a liquid from a reagent bottle to thesupplying pipette, a discharge pipette for discharging a liquid from thetubes, a discharge pump for suctioning drainage from the dischargepipette and discharging it to a drainage tank, and an agitatingmechanism having an up-down agitating mechanism for holding each tubeand moving it up and down and a driving unit for axially rotating theoscillating buckets in clockwise and anticlockwise directions; and aXYZ-direction movable pipetting mechanism that allows the analyteharvested by using the centrifuge to be dropped on a glass slide mountedon the thermostatic block.
 9. A device for preparing a sample slideaccording to claim 6 or 7, further comprising: a saturated moisturevalue detecting unit for outputting a saturated moisture value at atemperature detected by the temperature sensor by referring to asaturated moisture value in the same condition stored in a memory; adryness calculating unit for outputting dryness determined according tothe following equation using the saturated moisture value determined bythe saturated moisture value detecting unit and an actual absolutehumidity measured by the humidity sensor:Dryness (Idry)=Saturated moisture (Ws)−Absolute humidity (AbsH).
 10. Adevice for preparing a sample slide according to claim 8, furthercomprising: a saturated moisture value detecting unit for outputting asaturated moisture value at a temperature detected by the temperaturesensor by referring to a saturated moisture value in the same conditionstored in a memory; a dryness calculating unit for outputting drynessdetermined according to the following equation using the saturatedmoisture value determined by the saturated moisture value detecting unitand an actual absolute humidity measured by the humidity sensor:Dryness (Idry)=Saturated moisture (Ws)−Absolute humidity (AbsH).